This invention relates to a novel chemokine-binding protein and chemokine-binding fragments thereof. In particular, the invention relates to uses of the protein or fragments in a medicament, for example as an anti-viral or an anti-inflammatory agent; and to methods for the detection and inhibition of chemokines.
Chemokines are a family of small secreted polypeptides that regulate trafficking and effector functions of leukocytes, and play an important role in inflammation and host defence against pathogens (D'Souza & Harden, 1996; Fauci, 1996; Howard et al., 1996; Murphy, 1996; Premack & Schall, 1996; Baggiolini et al., 1997). More than 30 chemokines have been identified and these are divided into at least three structural groups based on the number and arrangement of conserved cysteines: CC (β) chemokines such as RANTES (for regulation-upon-activation, normal T cell expressed and secreted) and macrophage inflammatory protein (MIP)-1α, CXC (α) chemokines such as interleukin-8 (IL-8) and growth-related oncogene (GRO)-α, and the C chemokine lymphotactin. The different chemokines have evolved to function with particular cell types: many CC chemokines are chemoattractants for monocytes or thymocytes, while many but not all CXC chemokines are chemoattractants for neutrophils. However, there is no rule relating chemokine structure and cellular specificity to other leukocyte subtypes. For example, the C chemokine lymphotactin is selective for T cells, the CC chemokine eotaxin is specific for eosinophils and the CXC chemokines IFN-γ-induced protein (IP-10) and monokine induced by IFN-γ (Mig) attract activated T cells.
Chemokine receptors (CKRs) are seven transmembrane domain proteins and are coupled to G proteins for signal transduction. Most CXC chemokines have high affinity for only a single CKR (IL-8 is an exception in binding to CXCR1 and CXCR2), whereas most CC chemokines bind to more than one CKR (Baggiolini et al., 1997).
The activity of chemokines is tightly regulated to prevent excessive inflammation that can cause disease. Inhibition of chemokines by neutralising antibodies in animal models (Sekido et al., 1993) or disruption of mouse chemokine genes (Cook et al., 1995) have confirmed a critical role of chemokines in vivo in inflammation mediated by virus infection or other processes. The production of soluble versions of cytokine receptors containing only the extracellular binding domain, represents a physiological and therapeutic strategy to blockade the activity of some cytokines (Rose-John & Heinrich, 1994). However, the seven transmembrane domain structure of CKRs makes the construction of soluble, inhibitory CKRs difficult, and thus mutated chemokine antagonists, blocking peptides or antibodies are alternative inhibitors of chemokines under evaluation (D'Souza & Harden, 1996; Howard et al., 1996).
CKRs play a critical role in transmission and dissemination of HIV by acting as a cofactor which is required together with CD4 for virus entry and infection (D'Souza & Harden, 1996; Fauci, 1996). The CXCR4 CKR is a cofactor for T cell line-tropic HIV isolates, whereas the CCR5 (and CCR3) CKRs are involved in infection by macrophage-tropic HIV strains. The importance of CCR5 in vivo is supported by the finding that individuals who are homozygous for a mutant version of the CCR5 gene are resistant to HIV infection. Binding of chemokines or mutated chemokine antagonists to CKRs block HIV infection, illustrating the potential of the blockade of HIV-CKR interaction as a preventive and therapeutic strategy against HIV (D'Souza & Harden, 1996; Fauci, 1996).
Poxviruses are a group of large DNA viruses that replicate in the cytoplasm of the cell and encode many of their own enzymes for transcription and DNA replication (Moss, 1996). Vaccinia virus (VV) is the most intensively studied poxvirus and is famous as the live vaccine that was used to eradicate smallpox (Fenner et al., 1988). Since 1982 VV has also been widely used as an expression vector and in vaccine research (Moss, 1991). The genome of VV strain Copenhagen has been completely sequenced and contains approximately 200 genes (Goebel et al., 1990). Those near the centre of the genome are mostly highly conserved between poxviruses and many are essential for virus replication. In contrast, genes located towards either end of the genome are more variable between different viruses and are frequently non-essential for virus replication (Johnson et al., 1993). A subset of these non-essential virus genes are important for blocking specific components of the host immune system and many affect virus virulence. Thus VV and other poxviruses express proteins that are able to block the action of interferons, complement, cytokines, chemokines, inflammation and fever (Alcami & Smith, 1995a; McFadden et al., 1995; Smith, 1996; Spriggs, 1996; Smith et al., 1997c). Many of these proteins are secreted from the infected cell and bind to host factors in solution or at the cell surface. In many cases there is amino acid similarity between the virus protein and the extra cellular ligand-binding domain of a cell surface receptor for a particular ligand. In these cases it seems likely that the virus gene has been derived from the host during evolution.
Soluble chemokine-binding proteins have been previously detected in poxviruses. Firstly, the myxoma virus T7 protein, which was first identified as a soluble IFN-γR (Upton et al., 1992), binds to a range of chemokines through the heparin-binding domain and affects the infiltration of cells into infected tissue (Mossman et al., 1996; Lalani et al., 1997). The protein is described in WO 96/33730, designated CBP-1. In contrast, the IFN-γR from orthopoxviruses such as VV does not bind chemokines. (Alcami et al., 1998). Secondly, it was demonstrated that VV strain Lister expresses a soluble 35 kDa protein that is secreted from infected cells and which binds many CC chemokines (Graham et al., 1997; Smith et al.,1997a; Smith et al., 1997b; Alcami et al., 1998), but not CXC chemokines, through a domain distinct from the heparin-binding domain (Smith et al.,1997a; Smith et al., 1997b; Alcami et al., 1998). This protein has been called vCKBP (Alcami et al., 1998). The protein is also described in WO97/11714, as p35. Very similar proteins are made by some but not all VV strains, cowpox virus, the leporipoxviruses, Shope fibroma virus (SFV) and myxoma virus (called T1 protein), and by variola major viruses, e.g. protein G5R in India-1967 (Shchelkunov et al., 1994). Another VV gene encodes a protein more distantly related to vCKBP. In VV strain Copenhagen this was called A41L (Goebel et al., 1990), and in VV strain WR it was originally named SaIF4L (Howard et al., 1991) but renamed SaIF3L (Smith et al., 1991). Hereafter it is referred to as A41L. The relatedness of A41L and SFV T1, including the co-alignment of 8 cysteine residues was noted (Howard et al., 1991). Howard et al., 1991 also noted a potential site for attachment of carbohydrate via an asparagine residue and a putative signal peptide that might translocate the protein across the endoplasmic reticulum membrane and out of the cell. Other authors claimed there was no similarity between A41L and the 35 kDa protein of VV Lister and SFV T1 protein (Martinez-Pomares et al., 1995). In all three strains of variola major virus that have been sequenced there is an open reading frame (ORF) with more than 95% amino acid identity to the VV WR A41L protein termed 16L in strain Harvey (Aguado et al., 1992), A44L in strain Bangladesh-1975 (Massung et al., 1994) and A46L in strain India-1967 (Shcheikunov et al., 1994).
The similarity of A41L to vCKBP suggested that the A41L protein might bind chemokines as for vCKBP (Alcami et al., 1998). However Alcami et al., (1998) reported the supernatants of cells infected with VV WR or VV Lister from which the gene encoding the 35 kDa protein had been deleted (Patel et al., 1990), did not contain a protein that could be crosslinked to MIP-1α, RANTES, IL-8 or GRO-α (Alcami et al., 1998).
It has now been discovered that gene A41L encodes a 30 kDa protein that is secreted from cells infected by all strains of orthopoxvirus examined including 16 strains of VV and 2 strains of cowpox virus. In addition it is predicted to be expressed from all 3 strains of variola major virus for which sequence data are available (Aguado et al., 1992; Massung et al., 1994; Shchelkunov, 1995). The protein contains O- and N-linked carbohydrate. A VV WR deletion mutant lacking the A41L gene was constructed and shown to replicate in cell culture to the same titre as wild type and revertant viruses. Using A41L protein made by recombinant baculovirus in surface plasmon resonance (BIAcore), the protein was found to bind the CXC chemokines Mig and IP-10, but not other CXC, CC or C chemokines.
The invention provides in one aspect a medicament comprising an effective amount of an A41L protein or a chemokine-binding fragment thereof, together with a pharmaceutically acceptable carrier.
The invention is concerned in particular with the use of an A41L protein or a chemokine-binding fragment thereof as an anti-inflammatory agent. The A41L protein and fragments will be useful for the treatment of conditions mediated by chemokines, in particular CXC chemokines and IFN-γ-induced chemokines such as Mig and IP-10.
The invention is also concerned with the use of an A41L protein or a chemokine-binding fragment as an anti-viral agent. The A41L protein or fragment will inhibit binding to and/or infection of a target cell by pathogens such as HIV which mediate infection of a target cell via a cellular chemokine receptor.
The medicaments described herein may be useful for treatment and/or prophylaxis.
In another aspect, the invention provides a recombinant poxvirus which is genetically engineered to be incapable of expressing a native A41L protein.
Preferably, the recombinant poxvirus is incapable of expressing a chemokine-binding A41L protein, for example by deletion of all or a substantial part of the A41L gene. The recombinant poxvirus maybe for example a genetically engineered vaccinia virus. Vaccinia virus MVA, which is currently being developed for use in vaccines, is of particular interest in connection with this aspect of the invention. MVA genetically engineered so as to be incapable of expressing a chemokine-binding A41L protein has the anticipated advantages of being safer and less immunogenic than its counterpart expressing native A41L protein.
In a further aspect, the invention provides a detection method comprising the steps of:
a) providing a chemokine-binding molecule which is an A41L protein or a chemokine-binding fragment thereof;
b) contacting the chemokine-binding molecule with a sample;
c) detecting an interaction of the chemokine-binding molecule with a chemokine in the sample.
Thus, the invention may be used to detect the presence of one or more chemokines in a biological sample. The chemokine-binding molecule may be usefully immobilised on a solid support.
In another aspect, the invention provides a method for inhibiting the activity of one or more chemokines in a sample, which method comprises contacting the sample with an effective amount of a chemokine-binding molecule which is an A41L protein or a chemokine-binding fragment thereof.
In further aspects the invention provides a purified A41L protein or a chemokine-binding fragment thereof, for use in a method or a medicament as described herein; and a kit comprising such a purified A41L protein or fragment.
An amino acid sequence for the A41L protein is shown in FIG. 1. The sequence shown is for the VV WR A41L protein (accession no. D11079). Also shown for comparison is the amino acid sequence of vCKBP from VV Lister (DNA database of Japan, accession no. D00612).
The invention is concerned with A41L proteins (now also referred to as vCKBP-2) from poxviruses including vaccinia viruses and variola major viruses and from any other viral or non-viral source. Viral A41L proteins may be expected to have about 50% or more amino identity with the A41L amino acid sequence shown in FIG. 1, or particularly 80% or more, or 90% or more, or 95% or more amino acid identity with the A41L sequence in FIG. 1. Cellular homologues of the A41L protein which have the same or similar chemokine-binding capability may be expected to have a far lower amino acid identity with the sequence shown in FIG. 1, for example about 25%.
A41L proteins from different organisms may also be identified by the following shared characteristics.                conserved cysteine residues in the amino acid sequence which form intramolecular disulphide bonds.        chemokine-binding properties.        a similar size.        amino acid identity/similarity. Amino acid similarity means amino acid residues having similar properties such as charge and hydrophobicity; where one amino acid may be conservatively substituted (that is without significant effect on the structure of the protein) for another, there is “similarity” between the two.        acidity; the proteins are all reasonably acidic. This ties in with their function since chemokines tend to be basic proteins. The isoelectric point of the A41L protein from VV WR is 5.4        
The A41L proteins for use in the invention may be provided in native or in mutant form. Known methods, in particular genetic manipulation techniques, can be used to provide modified versions of the protein which have altered characteristics compared to the native protein. Desirable altered characteristics may be for example an altered binding capability to enhance binding to the chemokines.
The invention also envisages the use of fragments of the A41L protein, which fragments have chemokine-binding properties. The fragments may be peptides derived from the protein. Use of such peptides can be preferable to the use of an entire protein or a substantial part of a protein, for example because of the reduced immunogenicity of a peptide compared to a protein. Such peptides may be prepared by a variety of techniques including recombinant DNA techniques and synthetic chemical methods.
It will also be evident that the A41L proteins for use in the invention may be prepared in a variety of ways, in particular as recombinant proteins in a variety of expression systems. Such expression systems include for example poxvirus or non-poxvirus expression systems. Any standard systems may be used such as baculovirus expression systems or mammalian cell line expression systems.